Genetic Manipulation Technologies Research Articles

Utilizing machine learning and bioinformatics analysis to identify drought-responsive genes affecting yield in foxtail millet

Drought stress is a major constraint on crop development, potentially causing huge yield losses and threatening global food security. Improving Crop's stress tolerance is usually associated with a yield penalty. One way to balance yield and stress tolerance is modification specific gene by emerging precision genome editing technology. However, our knowledge of yield-related drought-tolerant genes is still limited. Foxtail millet (Setaria italica) has a remarkable tolerance to drought and is considered to be a model C4 crop that is easy to engineer. Here, we have identified 46 drought-responsive candidate genes by performing a machine learning-based transcriptome study on two drought-tolerant and two drought-sensitive foxtail millet cultivars. A total of 12 important drought-responsive genes were screened out by principal component analysis and confirmed experimentally by qPCR. Significantly, by investigating the haplotype of these genes based on 1844 germplasm resources, we found two genes (Seita.5G251300 and Seita.8G036300) exhibiting drought-tolerant haplotypes that possess an apparent advantage in 1000 grain weight and main panicle grain weight without penalty in grain weight per plant. These results demonstrate the potential of Seita.5G251300 and Seita.8G036300 for breeding drought-tolerant high-yielding foxtail millet. It provides important insights for the breeding of drought-tolerant high-yielding crop cultivars through genetic manipulation technology.

Molecular mechanisms that govern infection and antifungal resistance in Mucorales.

SUMMARYThe World Health Organization has established a fungal priority pathogens list that includes species critical or highly important to human health. Among them is the order Mucorales, a fungal group comprising at least 39 species responsible for the life-threatening infection known as mucormycosis. Despite the continuous rise in cases and the poor prognosis due to innate resistance to most antifungal drugs used in the clinic, Mucorales has received limited attention, partly because of the difficulties in performing genetic manipulations. The COVID-19 pandemic has further escalated cases, with some patients experiencing the COVID-19-associated mucormycosis, highlighting the urgent need to increase knowledge about these fungi. This review addresses significant challenges in treating the disease, including delayed and poor diagnosis, the lack of accurate global incidence estimation, and the limited treatment options. Furthermore, it focuses on the most recent discoveries regarding the mechanisms and genes involved in the development of the disease, antifungal resistance, and the host defense response. Substantial advancements have been made in identifying key fungal genes responsible for invasion and tissue damage, host receptors exploited by the fungus to invade tissues, and mechanisms of antifungal resistance. This knowledge is expected to pave the way for the development of new antifungals to combat mucormycosis. In addition, we anticipate significant progress in characterizing Mucorales biology, particularly the mechanisms involved in pathogenesis and antifungal resistance, with the possibilities offered by CRISPR-Cas9 technology for genetic manipulation of the previously intractable Mucorales species.

Genetic manipulation of betta fish.

Betta splendens, also known as Siamese fighting fish or "betta," is a freshwater fish species renowned for its astonishing morphological diversity and extreme aggressive behavior. Despite recent advances in our understanding of the genetics and neurobiology of betta, the lack of tools to manipulate their genome has hindered progress at functional and mechanistic levels. In this study, we outline the use of three genetic manipulation technologies, which we have optimized for use in betta: CRISPR/Cas9-mediated knockout, CRISPR/Cas9-mediated knockin, and Tol2-mediated transgenesis. We knocked out three genes: alkal2l, bco1l, and mitfa, and analyzed their effects on viability and pigmentation. Furthermore, we knocked in a fluorescent protein into the mitfa locus, a proof-of-principle experiment of this powerful technology in betta. Finally, we used Tol2-mediated transgenesis to create fish with ubiquitous expression of GFP, and then developed a bicistronic plasmid with heart-specific expression of a red fluorescent protein to serve as a visible marker of successful transgenesis. Our work highlights the potential for the genetic manipulation of betta, providing valuable resources for the effective use of genetic tools in this animal model.

Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Stable Transgenic Mouse Strain with Enhanced Photoactivatable Cre Recombinase for Spatiotemporal Genome Manipulation.

Optogenetic genome engineering is a powerful technology for high-resolution spatiotemporal genetic manipulation, especially for in vivo studies. It is difficult to generate stable transgenic animals carrying a tightly regulated optogenetic system, as its long-term expression induces high background activity. Here, the generation of an enhanced photoactivatable Cre recombinase (ePA-Cre) transgenic mouse strain with stringent light responsiveness and high recombination efficiency is reported. Through serial optimization, ePA-Cre is developed to generate a transgenic mouse line that exhibits 175-fold induction upon illumination. Efficient light-dependent recombination is detected in embryos and various adult tissues of ePA-Cre mice crossed with the Ai14 tdTomato reporter. Importantly, no significant background Cre activity is detected in the tested tissues except the skin. Moreover, efficient light-inducible cell ablation is achieved in ePA-Cre mice crossed with Rosa26-LSL-DTA mice. In conclusion, ePA-Cre mice offer a tightly inducible, highly efficient, and spatiotemporal-specific genome engineering tool for multiple applications.

Recent advances in genetic technology development of oleaginous yeasts

As important chemical raw materials and potential nutritional supplements, microbial lipids play an important role in ensuring economic development, food security, and energy security. Compared with non-natural hosts, oleaginous yeasts exhibit obvious advantages in lipid yield and productivity and have great potential to be genetically engineered into an oil cell factory. The main bottleneck in the current oleaginous yeasts engineering is the lack of genetic manipulation tools. Fortunately, the rapid development of synthetic biology has provided numerous new approaches, resources, and ideas for the field. Most importantly, gene editing technology mediated by CRISPR/Cas systems has been successfully applied to some oleaginous yeasts, almost completely rewriting the development pattern of genetic manipulation technology applicable. This paper reviews recent progress in genetic technology with regard to oleaginous yeasts, with a special focus on transformation methods and genome editing tools, discussing the effects of some important genetic parts. KEY POINTS: •Contribution of microbiotechnology in food safety and biofuel by oleaginous yeasts. •Advancement of genetic manipulation and transformation for oleaginous yeasts.

Gene activation in Caenorhabditis elegans using the Campylobacter jejuni CRISPR-Cas9 feeding system.

Clustered regularly interspaced palindromic repeats-based activation system, a powerful genetic manipulation technology, can modulate endogenous gene transcription in various organisms through fusing nuclease-deficient Cas9 to transcriptional regulatory domains. At present, this clustered regularly interspaced palindromic repeats-based activation system has been applied to activate gene expression by microinjection manner in Caenorhabditis elegans. However, this complicated and time-consuming injection manner is not suitable for efficient and high-throughput gene regulation with clustered regularly interspaced palindromic repeats-Cas9 system. Here, we engineered a Campylobacter jejun clustered regularly interspaced palindromic repeats-Cas9-based gene activation system through bacteria feeding technique to delivering gene-specific sgRNA in C. elegans. It enables to activate various endogenous genes efficiently, as well as induce the corresponding phenotypes with a more efficient and labor-saving manner. Collectively, our results demonstrated that our novel dCjCas9-based activation feeding system holds great promise and potential in C. elegans.

Zebrafish: An Emerging Model for Studying Macrophage Functions in Cancer

Abstract Zebrafish provide a convenient and unique model for studying human cancers, owing to the high similarity between zebrafish and human genomes, the availability of genetic manipulation technologies, and the availability of large numbers and transparency of zebrafish embryos. Many researchers have recently used zebrafish cancer models to examine the functions of macrophages in tumorigenesis, tumor growth and metastasis. Here, we present evidence that zebrafish cancer cells produce signals that are conserved with respect to those in humans and lead to the recruitment of heterogeneously activated macrophages in response to specific tumor types and tumorigenic stages, thereby promoting cancer initiation and progression. We also summarize how cancer cells interact with macrophages, emphasizing live imaging studies for visualization of dynamic material interchange.

Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production.

Hericium erinaceus is an important medicinal fungus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more difficult because of the many genes involved in central metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with high production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation technology for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the function and activity of A6180. The recombinant overexpression vector pET22b-A6180 was constructed for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal conditions (pH 6.0, 30°C). In addition, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the protein coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results indicate that A6180 plays an important role in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with higher activity by introducing A6180 into polysaccharide-producing mushrooms.

Construction of genome-wide protein tagging cell and mouse libraries.

Mice are the most widely used model organism for the study of gene functions and disease mechanisms through the generation of gene-modified mice. Since the 1980s, different genetic manipulation technologies have been developed to reveal gene functions in vivo, including homologous recombination strategies mediated by embryonic stem cells, transgenic strategies mediated by gametes, and the latest genetic modification strategies based on CRISPR/Cas9 technology. Semi-cloning technology mediated by "artificial spermatids" (androgenetic haploid embryonic stem cells, also termed sperm-like stem cells) is developed by Chinese scientists in 2012. In combination with CRISPR/Cas9, semi-cloning technology enables one-step generation of gene-modified mice through injection of "artificial spermatids" with specific gene modifications into oocytes. It has the characteristics of short construction cycle, high efficiency, low cost, and high application compatibility. In 2017, the Center for Excellence in Molecular Cell Science (CEMCS) of CAS has launched the genome tagging project (GTP) based on "artificial spermatid"-mediated semi-cloning technology. The ambitious goal of GTP is to tag every protein in mice and construct a unique mouse library that maintains the genome-wide protein-tagging mouse models. Subsequently, the GTP center was established at CEMCS to pursue the project. GTP center developed strategies to generate protein-tagging cells and mice. Briefly, a tag sequence is precisely inserted in a specific protein- coding gene endogenously in cultured "artificial spermatids"in vitro to build a cell library, in which, each cell line carrying a specific protein tag. The tagged cells could be further used as a sperm replacement to produce tagged mice in one step upon injection into oocytes. The tagged mouse library enables global analysis of protein expression, localization, and complexes using standard tag-based assays in vivo. By April 2021, the GTP center has generated 1532 tagged cell lines, 277 of which have been successfully used to produce tagged mice through oocyte injection. A total of 242 tagged mouse strains have been distributed to 66 research teams in 32 research institutions of 15 districts in 3 countries. The database of tagging product resources has been established and released regularly on the GTP website for scientists to inquire and order. Later, more information about GTP products, such as mouse breeding, protein tissue expression map, published literature, etc., will also be successively published on the GTP website. The GTP center will provide a standardized platform for protein function research, which may dramatically promote the development of life science and clinical transformation.

Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum.

Corynebacterium glutamicum has been considered a promising synthetic biological platform for biomanufacturing and bioremediation. However, there are still some challenges in genetic manipulation of C. glutamicum. Recently, more and more genetic parts or elements (replicons, promoters, reporter genes, and selectable markers) have been mined, characterized, and applied. In addition, continuous improvement of classic molecular genetic manipulation techniques, such as allelic exchange via single/double-crossover, nuclease-mediated site-specific recombination, RecT-mediated single-chain recombination, actinophages integrase-mediated integration, and transposition mutation, has accelerated the molecular study of C. glutamicum. More importantly, emerging gene editing tools based on the CRISPR/Cas system is revolutionarily rewriting the pattern of genetic manipulation technology development for C. glutamicum, which made gene reprogramming, such as insertion, deletion, replacement, and point mutation, much more efficient and simpler. This review summarized the recent progress in molecular genetic manipulation technology development of C. glutamicum and discussed the bottlenecks and perspectives for future research of C. glutamicum as a distinctive microbial chassis.

Establishment of a Cre-rat resource for creating conditional and physiological relevant models of human diseases

The goal of this study is to establish a Cre/loxP rat resource for conditional and physiologically predictive rat models of human diseases. The laboratory rat (R. norvegicus) is a central experimental animal in several fields of biomedical research, such as cardiovascular diseases, aging, infectious diseases, autoimmunity, cancer models, transplantation biology, inflammation, cancer risk assessment, industrial toxicology, pharmacology, behavioral and addiction studies, and neurobiology. Up till recently, the ability of creating genetically modified rats has been limited compared to that in the mouse mainly due to lack of genetic manipulation tools and technologies in the rat. Recent advances in nucleases, such as CRISPR/Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR associated protein 9), as well as TARGATT™ integrase system enables fast, efficient and site-specific introduction of exogenous genetic elements into the rat genome. Here, we report the generation of a collection of tissue-specific, inducible transgenic Cre rats as tool models using TARGATT™, CRISPR/Cas9 and random transgenic approach. More specifically, we generated Cre driver rat models that allow controlled gene expression or knockout (conditional models) both temporally and spatially through the Cre-ERT2/loxP system. A total of 10 Cre rat lines and one Cre reporter/test line were generated, including eight (8) Cre lines for neural specific and two (2) lines for cardiovascular specific Cre expression. All of these lines have been deposited with the Rat Resource and Research Center and provide a much-needed resource for the bio-medical community who employ rat models for their studies of human diseases.

Discovering the Molecular Determinants of Phaeobacter inhibens Susceptibility to Phaeobacter Phage MD18.

Bacteriophages have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. In this study, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and determined its mechanism of infection. Phaeobacter virus MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. The genomic sequence of MD18 displayed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8, but genomic and phylogenetic analyses, assessing host range and a search of available metagenomes are all consistent with the conclusion that Phaeobacter phage MD18 is a novel lytic phage. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, Gene Ontology (GO) term enrichment, and protein associations revealed that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens.IMPORTANCE Bacteriophages are useful nonantibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identified Phaeobacter virus MD18, a phage antagonist of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggested that Phaeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identified genes that modulate host susceptibility to phage MD18 and implicated the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses.

The misuse of genetic manipulation technology to enhance athletic performance is termed gene doping which is prohibited in human sports, horseracing, and equestrian sports. Although many qPCR assays have been developed, most assays employ genomic DNA (gDNA) from humans, non-human primates, and mice as a background and they may not be applicable for testing horse samples. This study aimed to develop a qPCR assay for the detection of human erythropoietin (hEPO) transgene in horse blood cells where the viral vectors used in gene therapy can reside for months. For the detection of hEPO transgene, the performance of three sets of primers and a hydrolysis probe for hEPO were compared. One set showed adequate specificity, sensitivity, amplification efficiency, and a dynamic range of detection in the presence of horse gDNA. The assay was duplexed with the detection of horse tubulin α 4A (TUBA4A) gene as an endogenous internal control in order to prevent false-negative results due to poor recovery and storage of extracted DNA and/or qPCR experimental variation. For the extraction of hEPO-plasmid, the QIAGEN Gentra Puregene blood kit was shown to recover the majority (62%) of hEPO-plasmid from spiked horse blood cells. The specificity and limit of detection (LOD) of the duplex qPCR assay were determined in accordance with MIQE guidelines. These findings supported the application of this duplex qPCR assay to the detection of hEPO transgene in horse blood cells.

Human papillomavirus 16 (HPV 16) E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by downregulating the expression of KIF7.

BackgroundThe E6 and E7 proteins in human papillomavirus 16 (HPV 16) are the main oncogenes in the occurrence of lung cancer. In recent studies, we found that E6 and E7 downregulated the expression of LKB1 in lung cancer cells. However, it is still unclear how E6 and E7 regulate LKB1 in lung cancer cells.MethodsDouble directional genetic manipulation and nuclear plasma separation technology were performed to explore the molecular mechanism of E6 and E7 inhibiting the antitumor activity of LKB1 in well‐established lung cancer cell lines.ResultsE6 but not E7 significantly downregulated the expression of tumor suppressor KIF7 at protein level, and the inhibition of KIF7 further reduced the expression of LKB1 both in the nuclei and in the cytoplasm, whereas reduced the expression of p‐LKB1 in the cytoplasm only. This suggested that HPV 16 E6 but not E7 downregulates the antitumor activity of LKB1 by downregulating the expression of p‐LKB1 in the cytoplasm only.ConclusionsHere, we demonstrated for the first time that E6 but not E7 inhibits the antitumor activity of LKB1 in lung cancer cells by downregulating the expression of KIF7. Our findings provide new evidence to support the important role of KIF7 in the pathogenesis of lung cancer and suggests new therapeutic targets.

Studies on DNA-related enzymes to elucidate molecular mechanisms underlying genetic information processing and their application in genetic engineering.

Recombinant DNA technology, in which artificially "cut and pasted" DNA in vitro is introduced into living cells, contributed extensively to the rapid development of molecular biology over the past 5 decades since the latter half of the 20th century. Although the original technology required special experiences and skills, the development of polymerase chain reaction (PCR) has greatly eased in vitro genetic manipulation for various experimental methods. The current development of a simple genome-editing technique using CRISPR-Cas9 gave great impetus to molecular biology. Genome editing is a major technique for elucidating the functions of many unknown genes. Genetic manipulation technologies rely on enzymes that act on DNA. It involves artificially synthesizing, cleaving, and ligating DNA strands by making good use of DNA-related enzymes present in organisms to maintain their life activities. In this review, I focus on key enzymes involved in the development of genetic manipulation technologies.

CRISPR/Cas with ribonucleoprotein complexes and transiently selected telomere vectors allows highly efficient marker-free and multiple genome editing in Botrytis cinerea.

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.

Can reproductive genetic manipulation save lives?

It has recently been argued that reproductive genetic manipulation technologies like mitochondrial replacement and germline CRISPR modifications cannot be said to save anyone's life because, counterfactually, no one would suffer more or die sooner absent the intervention. The present article argues that, on the contrary, reproductive genetic manipulations may be life-saving (and, from this, have therapeutic value) under an appropriate population health perspective. As such, popular reports of reproductive genetic manipulations potentially saving lives or preventing disease are not necessarily mistaken, though such terminology still requires further empirical validation.

Enhance transgene responses through improving cellular uptake and intracellular trafficking by bio-inspired non-viral vectors

BackgroundGene therapy remains a significant challenge due to lots of barriers limiting the genetic manipulation technologies. As for non-viral delivery vectors, they often suffer insufficient performance due to inadequate cellular uptake and gene degradation in endosome or lysosome. The importance of overcoming these conserved intracellular barriers is increasing as the delivery of genetic cargo.ResultsA surface-functionalized non-viral vector involving the biomimetic mannitol moiety is initiated, which can control the cellular uptake and promote the caveolae-mediated pathway and intracellular trafficking, thus avoiding acidic and enzymatic lysosomal degradation of loaded gene internalized by clathrin-mediated pathway. Different degrees of mannitol moiety are anchored onto the surface of the nanoparticles to form bio-inspired non-viral vectors and CaP-MA-40 exhibits remarkably high stability, negligible toxicity, and significantly enhanced transgene expression both in vitro and in vivo.ConclusionsThis strategy highlights a paradigmatic approach to construct vectors that need precise intracellular delivery for innovative applications.

How We Got to CRISPR: The Dilemma of Being Human.

This article considers the existential and eugenic risks of gene editing with CRISPR-Cas9. It brings forward epistemological and phenomenological questions concerning what CRISPR technology suggests about the limits of being human. By illuminating the paradoxical relationship between our "then self" and "now self," it considers the fragility of our individual and collective future-making endeavors. To do this, the article offers an overview of the existential dilemmas facing modern subjects, a history of eugenics and the ideology of health, a meditation on the limits of human knowledge, and an explication of the cultural work of metaphor. It argues for the benefits of human variation for individuals and communities and concludes by calling for an attitude of humility and restraint to guide the development and implementation of humane technologies rather than genetic manipulation technologies that aim to control future outcomes through present actions.